Bacteria can grow into surface-associated communities termed biofilms that are pervasive virtually everywhere on earth. [1] This mode of growth poses a significant obstacle to the successful treatment of infectious disease, with an estimated 80% of human infections in the biofilm state. [2] Biofilms are particularly problematic to clear due to their encasement in a protective and impermeable extracellular matrix, [3] which render biofilm-associated bacteria resistant to both host immune responses and standard antibiotic agents. Indeed, treatment with ˜10-1000-fold higher doses of antibiotic is often required for biofilm clearance relative to planktonic bacteria. [2] Biofilm growth by the Gram-negative bacterium Pseudomonas aeruginosa has attracted particular attention, as biofilms of this pathogen are the origin of the fatal chronic lung infections in most cystic fibrosis patients. [4] P. aeruginosa biofilm infections also plague burn victims, AIDS patients, and are endemic on the medical implants and devices universal in healthcare today. [5]
As such, the development of new methods to attenuate bacterial biofilm growth is of significant importance and represents a major research area. [6] Small molecules capable of inhibiting the growth of biofilm and particularly those capable of removing (i.e., dispersing) preformed biofilms would be extremely useful to combat bacterial infection in a range of applications in industry, agriculture, and the environment. [7]
Molecules exhibiting these properties however, remain rare. [5-14] This invention relates to a chemical approach for the inhibition and dispersion of bacterial biofilm, particularly those of Gram negative bacterial, more particularly those of Pseudomonas species and specifically those of P. aeruginosa, which is based on 2-aminobenzimidazoles.
Biofilm growth only occurs after a critical bacterial cell density is achieved, and in many bacteria is under the direct control of the cell-cell signaling pathway termed quorum sensing (QS). [6,7,15] Notably, P. aeruginosa mutants lacking a functional QS system are unable to grow into mature biofilms and are largely avirulent. [16,17] It has been shown, that non-native analogs of natural N-acylated L-homoserine lactone (AHL) QS signals can strongly modulate QS in Gram-negative bacteria, [18] and several of these AHLs also attenuate biofilm growth in P. aeruginosa. [11] One challenge to the application of AHLs as biofilm or QS inhibitors, however, is the hydrolytic instability of the lactone head group. [19] Hydrolyzed AHLs are biologically inactive, and therefore additional measures (e.g., multiple dosing, controlled delivery, etc.) are required for sustained activity of AHLs. [20, 21] Furthermore, and of particular relevance to biofilms, many AHL-derived biofilm inhibitors failed to disperse preformed biofilms, similar to most antibiotics. [11] In this context, there is a significant need in the art to identify hydrolytically stable molecular scaffolds for QS and/or biofilm modulation with enhanced activities.
Bacterial quorum sensing systems comprise small molecules, e.g., certain acyl-homoserine lactones, to regulate, in a cell-density dependent manner, a wide variety of physiological processes unique to the life-cycle of each microbe. These processes, which are collective designated “symbiotic behavior” herein, include: swarming, motility, sporulation, biofilm formation, conjugation, bioluminescence and/or production of pigments, antibiotics and enzymes.
A few natural products have been reported to inhibit bacterial QS or biofilm growth. The halogenated furanones from the macroalga Delisea pulchra have seen the most intensive study in this regard. [9] Three other notable examples include bromoageliferin, 3-indolylacetonitrile, and resveratrol (Scheme 1). Bromoageliferin displays anti-biofilm activity in the Gram-negative bacterium Rhodospirillum salexigens, and recently Melander and co-workers have reported simplified analogs of this marine natural product with anti-biofilm activities, most notably, certain 2-aminoimidazole (2-AI) derivatives in Gram-negative bacteria [8] and 5-amido or 5-imido 2-aminobenzimidazole (2-ABI) derivatives in Gram-positive bacteria. [22] The plant auxin 3-indolylacetonitrile was reported to inhibit the formation of P. aeruginosa biofilms via a QS-dependent mechanism, [23] and the phytoalexin resveratrol [24] and related stilbene derivatives [25] have recently been reported to inhibit the LuxR-type QS receptors in Gram-negative bacteria.

Published PCT application, WO 2010/144686 (Melander et al.) reports the inhibition of P. aeruginosa and Acinetobacter baumannii biofilm formation by certain 2-aminobenzimidazole (2-ABI) derivatives. Specifically tested compounds are listed in Scheme 1 therein (compounds 2-16, all of which carry a substituent bonded to the benzimidazole ring with a C—N bond. This PCT application is incorporated by reference herein. These 2-ABI derivatives are reported not to inhibit biofilm formation of the tested Gram negative bacteria at 100 microM concentration. In contrast, these 2-ABI derivatives are reported to inhibit biofilm development at a concentration of 100 microM of at least two of the Gram-positive bacteria: MRSA (presumably Methicillin-resistant Staphylococcus aureus), vancomycin-resistant Enterococcus faecium (VRE), or Staphylococcus epidermidis. In Table 1, therein, IC50 and EC50 values are provided corresponding to the concentration of compound that inhibits 50% biofilm development and the concentration that disperses 50%, respectively, of a pre-formed biofilm of one of the listed Gram-positive bacteria. The data in Table 1 therein are incorporated by reference herein. Compound 3 therein is reported to exhibit the best activity profile. The representative biofilm inhibitor, compound 3, is also asserted to operate via a Zn(II)-dependent mechanism. The application further reports that two control compounds (17 and 18, therein) did not inhibit MRSA, VRE or S. epidermidis biofilm formation at 100 microM.

The 2-AB1 molecules (presumably of Table 1) are asserted to be “some of the most potent anti-biofilm agents identified to date that do not operate through a microbiocidal mechanism.” This reference is incorporated by reference herein in its entirety for descriptions of experiments performed and materials employed in such experiments.
US published application 2008/0181923 (Melander et al.) relates to various imidazole based compounds. Specifically tested compounds, listed at least in part in Table 1, therein, are reported to inhibit biofilm formation of listed bacteria, including Pseudomonas aeruginosa. This reference is incorporated by reference herein in its entirety for descriptions of experiments performed and materials employed in such experiments.
Published PCT application 2004/047769 relates to certain benzimidazoles and analogs thereof which are reported to possess antibacterial activity. Compounds were reported evaluated for in vitro antibacterial activity against S. aureus and E. coli (see Tables, therein). This reference is incorporated by reference herein in its entirety for descriptions of compounds prepared, methods of synthesis, antibacterial activity experiments performed and materials employed in such experiments.